A Flatworm to the Rescue

A Flatworm to the Rescue

The ion channel TRPA1 is in some respects a conundrum, particularly regarding its contribution to temperature sensing. Some variants of TRPA1 respond to heat, while others respond to cold or are even insensitive to temperature. Now, researchers led by Marco Gallio, Northwestern University, Evanston, US, look to a simple creature to illuminate this intricate function of the channel.

The authors use RNA interference technology (RNAi) to show that TRPA1 present in the freshwater planarian S. mediterranea—a humble flatworm—is necessary for noxious heat avoidance, and for chemical nociception. They also show that planarian TRPA1 or even human TRPA1, neither of which respond directly to heat, could rescue heat sensing in Drosophila mutants missing the channel. Further, the investigators identify activation of TRPA1 by reactive oxygen species as a mechanism by which the channel regulates the response to harmful heat. Together, the results reveal the ancient evolutionary workings of TRPA1-mediated temperature sensation in diverse animals and show that noxious heat sensing by the channel depends on more than just direct heat gating.

“I really like this paper. It ties together a lot of interesting things about TRPA1 and delves into the evolutionary importance of the channel,” says Lindsey Macpherson, University of Texas at San Antonio, US, who was not involved in the study. “That planarians have a version of TRPA1 that responds to noxious heat is new and interesting, but the paper goes beyond that to get toward a mechanism of how TRPA1 is activated. It’s a fascinating look at TRPA1 through multiple lenses.” Macpherson’s previous work, undertaken as a graduate student in the lab of Ardem Patapoutian, Scripps Research Institute, La Jolla, US, revealed that pungent compounds such as mustard oil activate TRPA1 by binding to cysteines within the channel, a mechanism also identified at the same time by David Julius and colleagues (Macpherson et al., 2007; Hinman et al., 2006).

The new study was published online October 16 in Nature Neuroscience.

Why flatworms?

Gallio’s group studies the representation of temperature in Drosophila. The group's previous work revealed that these flies possess sensory neurons that respond to heating or cooling (Gallio et al., 2011; Frank et al., 2015). Research from other groups has shown the flies have a system to detect noxious heat that relies upon TRPA1 (Hamada et al., 2008; Neely et al., 2011). Nonetheless, the channel’s role in mediating temperature sensation is perplexing.

“Our lab and other labs have made puzzling observations on the temperature activation of TRPA1,” Gallio told PRF. For instance, “TRPA1 channels from humans and mice are supposedly activated by cold, while in flies and many other insects TRPA1 can be activated by heat. Snake TRPA1 is activated by heat, unlike TRPA1 in other vertebrates. So the activation by temperature seems to have changed many times during evolution,” he said.

Thus, Gallio decided to think “outside the box,” as he describes it, by turning to planarians. He thought studying flatworms could expand knowledge of temperature sensing, an area of research that has been dominated by studies in vertebrates, fruit flies, and the roundworm C. elegans.

“There are many animal phyla for which we know very little, particularly in neuroscience. That’s why we chose planarians, because they are in a phylogenetic position that makes them extremely distant from flies, C. elegans, and humans,” he said.

Another reason to use flatworms is that they actually engage in measurable behaviors. “It was exciting for us that planarians behave—they report what they like and what they don’t in terms of temperature. They move around, and we can subject them to the same assays for heat and cold avoidance that we use for flies,” according to Gallio.

Escaping the heat

First author Oscar Arenas and colleagues began by cloning a full-length coding sequence for the S. mediterranea TRPA1 gene, which they called Smed-TRPA1. To test the role of Smed-TRPA1 in noxious heat avoidance, the researchers used a two-choice avoidance assay similar to one they had previously developed for use in their Drosophila studies. In this case, the worms are placed in a circular chamber covered with a thin layer of water, and have to choose between staying on floor tiles held at a moderate (24° Celsius) temperature or on opposing floor tiles kept at a hot (32° Celsius) temperature.

The worms veered away from the hot quadrant when encountering the boundary between hot and cold, preferring to stay at the 24° Celsius temperature. But when RNAi was used to knockdown Smed-TRPA1, this heat avoidance disappeared. Now the worms spent nearly equal amounts of time in the hot and cool quadrants, swimming around in a nice circular pattern, compared to wild-type worms and control worms receiving double-stranded RNA that targets a sequence that the worm genome does not contain.

Smed-TRPA1 not only mediated noxious heat avoidance, but chemical nociception, too. To demonstrate this, the researchers used an assay consisting of two circular chambers connected by a corridor down which the worms are reluctant to pass. The team then placed either a mock agar pellet or an agar pellet also containing allyl isothiocyanate (AITC), the latter a TRPA1 agonist found in mustard and wasabi, into one of the chambers. Untreated worms or RNAi controls stayed near the mock pellets, but when exposed to the AITC pellets quickly turned away from them and then swam down the corridor. In contrast, this escape behavior was absent in Smed-TRPA1 knockdown worms, which remained near the AITC-containing pellets.

Across-phylum rescue

Turning next to in vitro biophysical properties of planarian TRPA1, the researchers used whole-cell patch clamp electrophysiology in Drosophila cells in which Smed-TRPA1 had been heterologously expressed. Based on their RNAi knockdown experiments showing that planarian TRPA1 mediated noxious heat avoidance, the group thought the channel would be gated by heat, just like many insect TRPA1 channels are. But this was not the case: The channel responded to AITC, but not to heat, in the heterologous system.

While this observation appeared odd, previous research had shown there was a Drosophila TRPA1 channel variant that was also insensitive to heat, and that this version could actually rescue the heat phenotype of mutant flies missing TRPA1. As a result, Gallio wondered if he could also rescue the heat phenotype of these fly mutants using planarian TRPA1. And indeed they could, even though the planarian TRPA1 did not respond to heat in vitro and lives in a cold-water environment of a completely different temperature than what a fly is used to. In fact, even human TRPA1, which supposedly responds to cold and not heat, rescued the heat phenotype of the fly mutants.

How could flatworm or human TRPA1 channels, which are not directly activated by heat, rescue a heat phenotype in a TRPA1 mutant fly?

An explanation emerges

One possible answer, Gallio reasoned, might be reactive oxygen species (ROS). Previous studies had shown that hydrogen peroxide (H2O2), a ROS and early marker of tissue damage, can activate TRPA1. This led to a hypothesis: Perhaps noxious heat produces tissue damage, resulting in production of H2O2 and/or other ROS, which then activate TRPA1 to enable noxious heat avoidance.

Three lines of evidence from the current study supported this hypothesis. First, in vitro experiments showed that Smed-TRPA1, when again heterologously expressed in Drosophila cells, could be activated by H2O2, as could a heat-insensitive Drosophila TRPA1 variant.

Second, using a dye that fluoresces when ROS are around, the researchers saw that live planarians loaded with the dye quickly produced ROS when subjected to rapid heating; similar findings were observed in Drosophila.

Third, H2O2 production affected behavioral responses to noxious heat. Here, the investigators fed Drosophila H2O2 or paraquat (an insecticide that induces ROS) 20 minutes before testing the flies for heat avoidance in a two-choice assay. While wild-type and control flies now showed increased heat avoidance, that is, they were sensitized to heat, mutants missing TRPA1 did not. “We think that ROS activation of the channel might be a core conserved mechanism for heat nociception,” Gallio said.

“This really gets to the crux of the system,” Macpherson said, “where TRPA1 channels look like they are able to be activated by reactive oxygen species that are produced by heating.”

The ROS hypothesis also explains a remarkable finding from the group’s earlier rescue experiments. There, planarian or human TRPA1 restored the heat phenotype of the Drosophila mutants to the temperature at which the flies—not planarians or humans—avoid noxious heat.

“These channels in their normal cellular context work at very different temperatures, and yet they can rescue the phenotype of the fly to the fly heat preference. The temperature at which H202 is produced is determined by the biology of the animal, which would dictate when the thermal damage is done,” Gallio said. “So as long as the fly tissue produces H202 at that temperature, any TRPA1 would do the job,” Gallio said.

What are the implications?

Potentially, the new findings could clarify issues such as whether human TRPA1 is activated by noxious cold, which has been controversial. If noxious cold produces enough H2O2 and/or other ROS, this could activate the channel, an idea that has been suggested before (Miyake et al., 2016). “There are direct implications for how we think about the function of TRPA1 in pain transduction in humans,” Gallio said.

The study also presents intriguing possibilities to improve human health by targeting TRPA1 in other organisms. For example, maybe targeting the channel in Schistosoma, which are parasitic worms closely related to planarians, could make them uncomfortable enough to the point they no longer seek to infect humans. Likewise, Gallio says, possibly mosquito TRPA1 could be targeted. “Mosquitoes use heat to find human prey, and TRPA1 has been chosen as a candidate heat receptor to create mutant mosquitoes that would be heat blind. Our results suggest we should rather try to activate mosquito TRPA1” to prevent mosquitoes from having an interest in biting people (Corfas and Vosshall, 2015).

Perhaps ROS could also be targeted to control acute pain in people, for example. But this would become possible only if researchers discover ways to inhibit the channel.

“There are many activators of TRPA1, but we don’t have many inhibitors of the channel, so that’s the next step,” Macpherson told PRF.

A simple organism illuminates the function of a complex channel

In the meantime, Gallio is pursuing follow-up studies in planarians to understand whether the flatworms use similar or different systems to detect noxious versus non-noxious temperatures. He is doing such studies in Drosophila as well.

“Our results argue that the common ancestor of all bilateral animals probably had a cellular system to detect noxious stimuli that used TRPA1, one that worked using principles that are now broadly conserved in all animal groups. This scale of conservation is amazing, and we want to understand what else is conserved, and whether—like humans and flies—planarians may also possess a dedicated system for navigating innocuous hot and cold temperature,” Gallio said.

Ultimately, to Gallio, the new findings show that less complex creatures like flatworms have much to teach researchers about pain, an area of study that generally uses more complicated animals such as mice and humans.

“We still have a lot to learn from worms and flies in that they could be more approachable from an experimental point of view. They can help us understand the initial nociceptive signal, which is the first step toward pain in many cases.”